EP3487909A1 - A thermogelling supramolecular sponge as self-healing and biocompatible hydrogel - Google Patents
A thermogelling supramolecular sponge as self-healing and biocompatible hydrogelInfo
- Publication number
- EP3487909A1 EP3487909A1 EP17730074.6A EP17730074A EP3487909A1 EP 3487909 A1 EP3487909 A1 EP 3487909A1 EP 17730074 A EP17730074 A EP 17730074A EP 3487909 A1 EP3487909 A1 EP 3487909A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- block copolymer
- batch
- meox
- methyl
- copolymer according
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- IMSODMZESSGVBE-UHFFFAOYSA-N 2-Oxazoline Chemical compound C1CN=CO1 IMSODMZESSGVBE-UHFFFAOYSA-N 0.000 claims description 9
- 239000013543 active substance Substances 0.000 claims description 6
- BYVSMDBDTBXASR-UHFFFAOYSA-N 5,6-dihydro-4h-oxazine Chemical compound C1CON=CC1 BYVSMDBDTBXASR-UHFFFAOYSA-N 0.000 claims description 4
- 238000007334 copolymerization reaction Methods 0.000 claims description 4
- 238000012377 drug delivery Methods 0.000 claims description 3
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- OIRDBPQYVWXNSJ-UHFFFAOYSA-N methyl trifluoromethansulfonate Chemical compound COS(=O)(=O)C(F)(F)F OIRDBPQYVWXNSJ-UHFFFAOYSA-N 0.000 description 3
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- QTENRWWVYAAPBI-YZTFXSNBSA-N Streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@H]1[C@H](N=C(N)N)[C@@H](O)[C@H](N=C(N)N)[C@@H](O)[C@@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@H]1[C@H](N=C(N)N)[C@@H](O)[C@H](N=C(N)N)[C@@H](O)[C@@H]1O QTENRWWVYAAPBI-YZTFXSNBSA-N 0.000 description 1
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- INAHAJYZKVIDIZ-UHFFFAOYSA-N boron carbide Chemical compound B12B3B4C32B41 INAHAJYZKVIDIZ-UHFFFAOYSA-N 0.000 description 1
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- LDTLDBDUBGAEDT-UHFFFAOYSA-N methyl 3-sulfanylpropanoate Chemical compound COC(=O)CCS LDTLDBDUBGAEDT-UHFFFAOYSA-N 0.000 description 1
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- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
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- GLGXXYFYZWQGEL-UHFFFAOYSA-M potassium;trifluoromethanesulfonate Chemical compound [K+].[O-]S(=O)(=O)C(F)(F)F GLGXXYFYZWQGEL-UHFFFAOYSA-M 0.000 description 1
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- CWXPZXBSDSIRCS-UHFFFAOYSA-N tert-butyl piperazine-1-carboxylate Chemical compound CC(C)(C)OC(=O)N1CCNCC1 CWXPZXBSDSIRCS-UHFFFAOYSA-N 0.000 description 1
- GCSSSCOWYZYEFZ-UHFFFAOYSA-N tert-butyl piperazine-1-carboxylate Chemical compound CC(C)(C)OC(=O)N1CCNCC1.CC(C)(C)OC(=O)N1CCNCC1 GCSSSCOWYZYEFZ-UHFFFAOYSA-N 0.000 description 1
- 125000003831 tetrazolyl group Chemical group 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical class CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
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- YZYKBQUWMPUVEN-UHFFFAOYSA-N zafuleptine Chemical compound OC(=O)CCCCCC(C(C)C)NCC1=CC=C(F)C=C1 YZYKBQUWMPUVEN-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
- C08G73/02—Polyamines
- C08G73/0233—Polyamines derived from (poly)oxazolines, (poly)oxazines or having pendant acyl groups
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/075—Macromolecular gels
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2379/00—Characterised by the use of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing nitrogen with or without oxygen, or carbon only, not provided for in groups C08J2361/00 - C08J2377/00
- C08J2379/02—Polyamines
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2379/00—Characterised by the use of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing nitrogen with or without oxygen, or carbon only, not provided for in groups C08J2361/00 - C08J2377/00
- C08J2379/04—Polycondensates having nitrogen-containing heterocyclic rings in the main chain; Polyhydrazides; Polyamide acids or similar polyimide precursors
Definitions
- thermogelling supramolecular sponge as self-healing and biocompatible hydrogel
- Biocompatible polymers that form thermoreversible supramolecular hydrogels have gained great interest recently as so-called bioinks for 3D bioprinting in tissue engineering and biofabrication.
- thermogelling polymers find application in food and pharmaceutical technology, biology and medicine (J. D. Kretlow, S. Young, L. Klouda, M. Wong, A.
- thermoresponsive polymers are polymers obtained from cyclic imino ethers, particularly poly(2-substituted-2-oxazoline)s (POx) and poly(2-substituted-5,6-dihydro-4H-1 ,3-oxazine)s (in short poly(2-oxazine)s; POzi). These polymers are accessible via living cationic ring-opening polymerization (K. Aoi, M. Okada, Prog. Polym. Sci. 1996, 21, 151 -208) and can exhibit LCST in aqueous solution where the transition temperature can be tuned over a large tern- perature range (S. Huber, R. Jordan, Colloid Polym. Sci.
- thermoresponsive material C. Weber, R. Hoogenboom, U. S. Schubert, Prog. Polym. Sci. 2012, 37, 686-714; R. Hoogenboom, H. Schlaad, Po/ymere 201 1 , 3, 467-488; J.-H. Kim, Y. Jung, D. Lee, W.-D. Jang, Adv. Mater. 2016), but also for biomedical applications (A. C. Rinkenauer, L. Tauhardt, F. Wendler, K. Kempe, M. Gottschaldt, A. Traeger, U. S. Schubert, Macromol. Biosci. 2015, 15, 414-425; Z.
- thermoresponsive gels are currently heavily investigated, but new materials that allow the tuning of the response temperature and the rheological properties are urgently needed.
- thermogelling and rheological properties due to their specific structural composition.
- a block copolymer characterized by one of the general chemical structures [A] n -[B] m or [B] n -[A] m , wherein [A] is a poly(2-oxazine), and wherein [B] is a poly(2-oxazoline), wherein n and m are in the range of 20 to 300 each, and wherein n and m approximately have the same value.
- the block copolymer with regard to the present invention consists of at least two different blocks [A] and [B] with n or m repetition units each.
- a block copolymer is preferred, which is characterized by one of the general chemical structures [A] n -[B] m or [B] n -[A] m , wherein [A] is a poly(2-oxazine), and wherein [B] is a poly(2-oxazoline), wherein n and m are in the range of 40 to 300 each, and wherein n and m approximately have the same value.
- thermogelling block copolymers comprising a
- thermoresponsive poly(2-oxazine)s ((POzi)-block) and a hydrophilic poly(2- oxazoline)s ((POx)-block) were synthesized.
- the rheological properties of aqueous solution of these block copolymers were investigated by viscosimetry and rhe- ology. Excellent cytocompatibility was shown using NIH 3T3 fibroblasts. Therefore, these novel materials encompass all necessary parameters for use as bioink.
- the solution while liquid at 4 °C, solidified at the elevated temperature (> 25 °C) in laboratory. This is the first case of thermogelling polymers comprising solely poly(cyclic imino ethers).
- the block copolymers according to the present invention undergo thermogelation in different temperature regions.
- the block copolymers according to the present invention undergo thermogelation above 10°C, particularly above 25 °C (for example block copolymers with the general chemical structure [A]i 0 o-[B]ioo or [B]ioo-[A]ioo)-
- the block copolymers according to the present invention undergo thermogelation above 30 °C, particularly above 35 °C (for example block copolymers with the general chemical structure [A] 5 o-[B] 5 o or [B] 5 o-[A] 5 o).
- All block copolymers form transparent hydrogels of surprisingly high strength (G * > 1000 Pa) and show excellent and rapid shear recovery after stress.
- the new optical transparent gels have a very suitable and adjustable gelation temperature.
- the synthesis of the polymers is easy and scalable well. The gelation process is quite fast.
- the material is highly cytocompatible, gives relatively stable hydrogels (G' « 4 kPa), but shows pronounced shear thinning. As the formed hydrogels are optically clear, they are suitable for light microscopy.
- block copolymer most simply refers to polymers of at least two different polymer blocks, wherein each polymer block comprises two or more adjacent units of the same kind.
- block copolymer is used herein in accordance with its established meaning in the art to refer to copolymers wherein repeating units of a defined type are organized in blocks [A] and [B].
- the block copolymers of the present invention are synthesized by a two-stage copolymerization of 2-oxazin and 2-oxazoline. Further preferred, the block copolymers of the present invention are synthesized by a two-stage copoly- merization of 2-oxazoline and 2-oxazin.
- the mechanism showing such a two-stage copolymerization of 2-oxazoline and 2-oxazin is presented in the following general scheme:
- Block A (2-oxazoline as shown in the scheme above) and block B (2-oxazin as shown in the scheme above) can also be interchanged without resulting in a change of mechanical and physico-chemical properties of the synthesized block copolymer.
- the rest R (of 2-oxazoline and 2-oxazin as well) is preferably an alkyl-group, but of course not limited to this.
- [A] is chosen of a group containing 2-n-propyl-2-oxazine, 2-cyclopropyl- 2-oxazine and 2-butyl-2-oxazine and [B] is chosen of a group containing 2-methyl- 2-oxazoline and 2-ethyl-2-oxazoline.
- [A] and [B] are not limited to the above mentioned molecules.
- the block copolymer is characterized by one of the following general chemical structures:
- n is in the range of 20 to 300, wherein m is in the range of 20 to 300, and wherein n and m have the same or approximately the same value.
- block copolymer is characterized by one of the following general chemical structures:
- n is in the range of 40 to 300, wherein m is in the range of 40 to 300, and wherein n and m have the same or approximately the same value.
- the end group R in the above shown general chemical structures is introduced by an initiation molecule, starting the polymerization.
- the final group or termination group R 1 is introduced by a termination molecule or termination agent (nucleo- phile), which stops the polymerization reaction.
- the initiation molecules as well as the termination molecules may be chosen of a variety of substances. Depending on the selected substances, so depending on the structure of the initiation molecules and the termination molecules, block copolymers with different groups R and R 1 are synthesized.
- R may be introduced by an initiation molecule chosen of a group containing strong Bronsted, Lewis acids or alkylating agents.
- initiation molecule chosen of a group containing strong Bronsted, Lewis acids or alkylating agents.
- R may be an alkyl group.
- R 1 may be introduced by a nucleophilic termination molecule.
- the nucleophilic termination molecule may be chosen of a group containing hydroxide, Ethyl 4-piperidinecarboxylate, tert-butyl-1 - piperazinecarboxylate (1 -Boc-Piperazine), 3-mercapto-propionic acid methyl ester, piperazine and its derivates.
- R 1 may be a Piperidin-group.
- the invention is not limited neither to the above specified initiation molecules nor to the above specified termination molecules. Rather, these molecules and the resulting groups R and R 1 shall not effect the gelling behavior of the synthesized block copolymers. In other words, the observed effects with regard to the present invention occur basically independent of the groups R and R 1 .
- the block copolymer is characterized by the following chemical structure, wherein n and m have a value of 50 each.
- the block copolymer is characterized by the following chemical structure,
- n and m have a value of 100 each.
- Methyl-P[nPrOzi 5 o-b-MeOx 50 ]-piperidine-4-carboxylic acid ethyl ester as well as Methyl-P[nPrOziioo-b-MeOx 100 ]-piperidine-4-carboxylic acid ethyl ester were synthesized by polymerization of 2-n-propyl-2-oxazine (nPrOzi, thermoresponsive POzi block) and 2-methyl-2-oxazoline (MeOx, hydrophilic POx block). The detailed mechanism of the synthesis of the block copolymers is shown later on.
- the chemical purity of block [A] and/or of block [B] of a block copolymer with one of the general chemical struc- tures [A]n-[B] m or [B] n -[A] m is at least 75 %, in particular 90 %.
- block [A] comprises at least 75% of a first 2-oxazoline and up to 25 % of a second 2-oxazoline or 2-oxazine and/or that block [B] comprise at least 75% of a first 2-oxazine and up to 25 % of another 2-oxazine or 2-oxazoline.
- the block copolymer is characterized in forming a thermoresponsive hydrogel.
- the aqueous solution of the block copolymer forms a thermoresponsive hydrogel.
- each block copolymer according to the present invention is characterized by the thermogelling behavior of its aqueous solutions. Gel formation of aqueous polymer solutions only occurs if the concentration exceeds a certain value. With increasing molecular weight, this concentration decreases. For block copolymers with lower molecular weight (as for batches P1 to P5 shown later on), the critical concentration is 20 wt.-% for higher molecular weight is slightly lower at 18 wt.-% (as for batches P1 a to P6a shown later on as well).
- the gelling temperature may differ.
- the block copolymers may gel at lower temperatures with increasing chain length, so with higher values of n and m.
- the block copolymers gel preferably at temperatures above 30 °C, particularly above 35 °C (as an example block copolymers with the general chemical structure [A] 5 o-[B] 5 o or [B] 5 o-[A] 5 o)- Further preferred, the block copolymers gel at temperatures above 10 °C, preferably above 25 °C (as an example block copolymers with the general chemical structure [A] 0 o-[B]ioo or [B] 0 o-[A]i 0 o)-
- the block copolymer with regard to the present invention is used as a carrier material for an active agent.
- the block copolymer is used as a carrier material, wherein the active agent is embedded in carrier material.
- the block copolymer is used as a carrier material for time-delayed disposal of the embedded active agent.
- the usage of the block copolymer as a carrier material in a drug delivery system is also favored.
- the use of the block copolymer as a carrier material for cells is preferred.
- thermogelling in combination with the pronounced isothermal shear-thinning is of advantage for any of these noted applications.
- hydrogels formed from aqueous solution of the polymers disclosed in the present invention are optically transparent, which is advantageous for said applications.
- Dul- becco's Modified Eagle Medium (DMEM), fluorescein di acetate (FDA) and propidium iodide (PI) were purchased from Sigma-Aldrich (Schnelldorf, Germany). Penicillin G and streptomycin solution was purchased from Biochrom AG (Berlin, Germany). Fetale bovine serum (FBS) was from Gibco (Darmstadt, Germany). 8-well LabTek chamber slides were from Nunc (Thermo Fisher Scientific, Schrö, Germany).
- 96-well plates and 100 mm culture dishes were used from Greiner Bio One (Frickenhausen, Germany).
- Water soluble tetrazolium (WST-1 ) was used from Rache (Basel, Switzerland).
- Methyl trifluoromethylsulfonate (MeOTf), 2-methyl-2- oxazoline (MeOx), benzonitrile (PhCN) and other solvents were dried by refluxing over CaH 2 under dry argon atmosphere and subsequent distillation prior to use.
- NMR spectra were recorded on a Bruker Fourier 300 ( H 300.12 MHz) at 298 K, Bruker BioSpin (Rheinstetten, Germany). The spectra were calibrated using the solvent signals (MeOD 3.31 ppm). Gel permeation chromatography (GPC) was performed on a Polymer Standard Service (PSS, Mainz, Germany) system (pump mod. 1260 infinity, R I -detector mod.
- IR spectra were recorded on Jasco (GroB-Umstadt, Germany) FT/I R -4100 equipped with an ATR-unit.
- Rheology experiments were performed using an Anton Paar (Ostfildern, Germany) Physica MGR 301 utilizing a plate-plate geometry (diameter 25 mm). The rheometer was equipped with a Peltier element. All polymer solutions used for rheology were prepared just before the measurement and were kept in the fridge at approx. 8 °C.
- Dialysis was performed using Spectra Por membranes with a molecular weight cutoff (MWCO) of 1 and 4 kDa obtained from neoLab (Heidelberg, Germany).
- MWCO molecular weight cutoff
- the detector is a 6 Li-glass detector with an active area of 60x60 cm 2 .
- the exposure times were 5, 15 and 35 minutes for the SDDs of 1 .61 , 7.61 and 19.61 m respectively.
- the sample was filled into a Hellma cuvette with a light path of 1 mm. This cuvette was placed into a Julabo temperature controlled oven. Dark current correction was carried out using boron carbide.
- Murine NIH 3T3 fibroblasts (ATCC-Number CRL-1658, ATCC, Manassas, VA) were maintained in 100 mm culture dishes in growth medium (DMEM containing 10 % heat inactivated FBS, 100 U/mL penicillin G and 100 ⁇ g/ ⁇ L streptomycin) at 37 °C and 5 % CO 2 .
- the Iyophiiized polymer was dissolved in growth medium at 30 wt.-%. 20.000 NIH 3T3 fibroblasts dispersed in media were incorporated into the polymer stock- solution by stricte mixing with an Eppendorf pipette on ice to yield a 100 ⁇ _ solution, in which the final polymer concentration was 25 wt.-%. The solution was subsequently added to one well of a preheated (37° C) 8-well LabTek chambers slide.
- the cells were subsequently analysed by flow cytometry on a FACS Calibur system.
- a 488 nm Laser was chosen with the emission channel FL2 (585 nm / ⁇ 21 nm) for PI or the emission channel FL I (530 nm / ⁇ 15 nm) for FDA, respectively (see Figures 18 and 19).
- a total number of 5000 events were counted with BD CellQuestTM Pro and the geometric mean fluorescence intensity was determined for each condition using Flowing Software (version 2.5.1 ; Turku Bioimaging).
- the cell pellet of NIH 3T3 fibroblasts was FDA-stained and 20.000 cells were incorporated into a 25 wt.-% polymer solution and added into 37 °C preheated 8-well LabTek chambers slides as described above. FDA stained cells were subsequently analyzed with a Zeiss Observer Z1 epi-fluorescence microscope (Zeiss, Oberkochen, Germany) equipped with a 37 °C incubation chamber. 3D stacks with 1 ⁇ z-stack intervals were taken. Acquired 3D Stacks were analyzed with the ZEN Imaging Software (Zeiss, Oberkochen, Germany).
- 2000 NIH 3T3 fibroblasts were seeded in growth medium in a 96-well-format and incubated overnight at 37 °C and 5 % CO 2 .
- Dilution concentrations of the 30 wt.-% polymer stock solution were prepared (final polymer concentrations: 10 wt.-%, 5 wt.-%, 1 wt.-%, and 0.02 wt.-%) in growth medium on ice and added to the cells.
- Cell growth was stimulated for 48 h at 37°C and 5% CO 2 .
- the cell medium was carefully exchanged and replaced by fresh growth medium.
- the cells were incubated with WST-1 for 3 h at 37 °C according to the manufacturer's instructions.
- the absorbance of the soluble formazan product was determined at 570 nm using a Spectramax 250 microplate reader from Molecular Devices (Sunnyvale, USA).
- 2-n-propyl-2-oxazine As an example, the synthesis of the monomer 2-n-propyl-2-oxazine (nPrOzi) is shown. 2-n-propyl-2-oxazine was synthesized by an adapted standard procedure (S. Sinnwell, H. Ritter, Macromol. Rapid Commun. 2006, 27, 1335- 1340), as shown in the following scheme:
- Zincacetate dihydrate (catalyst) was dissolved in propionitrile and 3-amino-1 - propanol was added dropwise at room temperature. The reaction mixture was stirred under reflux conditions for at least 24 h. Progression of the reaction was monitored by IR-spectroscopy. After total nitrile consumption the monomer was purified by fractional distillation under inert argon atmosphere to obtain a clear colorless liquid (yield: 363.5 g, 55.5 %).
- 2-methyl-2-oxazoline was bought with a purity of 99% and distilled before polymerization under reduced pressure on molecular sieve.
- the monomer concentration at the beginning of the reaction is typically laying around 3 mol/l.
- the chain termination is performed typically at 40 ° C and for 4 h at least.
- monofunctional termination agents typically three equiva- lents per initiator molecule are inserted. At least ten equivalents are used in the case of bifunctional termination agents, as for example piperazine.
- Methyl-Poly[nPrOzi 5 o-b-MeOx 50 ]-piperidine-4- carboxylic acid ethyl ester (and Methyl-Poly[nPrOzii 0 o-b-MeOx 100 ]-piperidine- 4-carboxylic acid ethyl ester as well) is explained in detail.
- Poly[nPrOzi 5 o-b-MeOx 50 ]-piperidine-4-carboxylic acid ethyl ester was synthesized by polymerization of 2-n-propyl-2-oxazine (nPrOzi, thermoresponsive POzi block) and 2-methyl-2-oxazoline (MeOx, hydrophilic POx block), as shown in the following mechanism.
- potassium carbonate 232 mg, 1 .68 mMol, 1 eq
- the solvent was removed at reduced pressure from the supernatant after centrifugation and the flask was placed in a vacuum drying oven at 40 °C and 20 mbar for 2 days.
- the product was dissolved in ultra-purified water dialyzed overnight using a membrane with a MWCO of 4kD and freeze-dried (yield: 14.3 g, 79 %).
- Figure 1 shows GPC traces for different batches of Methyl-P[nPrOzi 5 o-b- MeOx 50 ]-piperidine-4-carboxylic acid ethyl ester, shows the temperature dependent rheology analysis with storage modulus (C) and loss modulus (G”) for 20 wt.-% of Methyl-
- Figure 5 shows an 1 H-NMR spectra of batch P3 in methanol-d 4 at 298 K of
- Figure 6 shows an 1 H-NMR spectra of batch P4 in methanol-d 4 at 298 K of
- Figure 7 shows a 1 H-NMR spectra of batch P5 in methanol-d 4 at 298 of Me- thyl-P[nPrOzi 5 o-b-MeOx 50 ]-piperidine-4-carboxylic acid ethyl ester,
- Figure 8 shows an amplitude sweep used to determine the LVE-range at 10
- Figure 9 shows a flow curve at 37 °C for 20 wt.-% of Methyl-P[nPrOzi 50 -b- eOx 50 ]-piperidine-4-carboxylic acid ethyl ester,
- Figure 10 shows shear recovery at 37 °C for 20 wt.-% of Methyl-P[nPrOzi 50 -b-
- MeOx 50 ]-piperidine-4-carboxylic acid ethyl ester shows the temperature- and concentration-dependent viscosity of Methyl-P[nPrOzi 5 o-b-MeOx 50 ]-piperidine-4-carboxylic acid ethyl ester at different concentrations compared with the temperature- and concentration-dependent viscosity of the Poloaxamer F127 at 10 wt.-% ,
- Figure 12 shows temperature-dependent SANS scattering data of Methyl-
- Figure 13 shows representative fits for a bi-continuous sponge like structure for lowest temperature (at 6.9 °C) and highest temperature (39.7 °C) of Methyl-P[nPrOzi 5 o-b-MeOx 50 ]-piperidine-4-carboxylic acid ethyl ester at 20 wt.-%,
- Figure 14 shows resulting correlation length ⁇ and characteristic domain size d of Methyl-P[nPrOzi 50 -b-MeOx 50 ]-piperidine-4-carboxylic acid ethyl ester at 20 wt.-%,
- Figure 15 shows fits of selected datasets over the complete temperature range of Methyl-P[nPrOzi 5 o-b-MeOx 50 ]-pipendine-4-carboxylic acid ethyl ester at 20 wt.-%,
- Figure 16 shows the proliferation of NIH-3T3 cells in the presence of various polymer concentrations of Methyl-P[nPrOzi 5 o-b-MeOx 50 ]-piperidine-4- carboxylic acid ethyl ester as analyzed by WST-1 assay at 20 wt.-%,
- Figure 17 shows Cell viability of NIH 3T3 fibroblasts
- Figure 18 shows a flow cytometry analysis of Methyl-P[nPrOzi 5 o-b-
- Figure 19 shows a flow cytometry analysis of NIH 3T3 fibroblasts cultivated in Methyl-P[nPrOzi 5 o-b-MeOx 50 ]-piperidine-4-carboxylic acid ethyl ester at 20 wt.-%,
- Figure 20 shows a temperature-dependent rheological analysis of Methyl- P[nPrOzi ⁇ -b-MeOx ⁇ ]-piperidine-4-carboxylic acid ethyl ester (P2) at a concentration of 20 wt.-%,
- Figure 21 shows a phase diagram of aqueous solutions of Methyl-P[nPrOzi 50 - b-MeOx 50 ]-piperidine-4-carboxylic acid ethyl ester (P2) dependent on concentration and temperature
- Figure 22 shows the temperature dependent rheology with storage modulus (G') and loss modulus (G") for 20 wt.-% of Methyl-P[nPrOz 0 o-b- MeOx 100 ]-piperidine-4-carboxylic acid ethyl ester and Methyl- P[nPrOzi 0 o-b-MeOx 100 ]-tert-butyl-piperazine-1 -carboxylat,
- Figure 23 shows the temperature dependent rheology with storage modulus
- G' loss modulus
- G" loss modulus for 20 wt.-% of Propynyl-P[nPrOzi 00 -b- MeOx 100 ]-piperidine-4-carboxylic acid ethyl ester , Propynyl- P[nPrOzi 10 o-b-MeOx 100 ]-methyl 3-mercaptopropionate and Propynyl- P[nPrOzi 10 o-b-MeOx 100 ]-hydroxy,
- Figure 24 shows GPC traces of Methyl-P[nPrOzi 1 ⁇ -b-MeOx 100 ]-piperidine-4- carboxylic acid ethyl ester and Methyl-P[nPrOzii 0 o-b-MeOx 100 ]- tert- butyl-piperazine-1 -carboxylat,
- Figure 25 shows GPC traces of Propynyl-P[nPrOzii ⁇ -b-MeOx 100 ]-piperidine-4- carboxylic acid ethyl ester, Propynyl-P[nPrOzi 1 ⁇ -b-MeOx 100 ]- methyl 3-mercaptopropionate and Propynyl-P[nPrOziioo-b-MeOx 100 ]-hydroxy,
- Figure 26 shows an 1 H-NMR spectra of Methyl-P[nPrOzi 10 o-b-MeOx 100 ] ⁇
- Figure 27 shows an 1 H-NMR spectra of Methyl-P[nPrOzi 1 ⁇ -b-MeOx 100 ]-tert- butyl-piperazine-1 -carboxylat in methanol-d 4 at 298 K,
- Figure 28 shows an 1 H-NMR spectra of Propynyl-P[nPrOzi 10 o-b-MeOx 100 ]- piperidine-4-carboxylic acid ethyl ester in methanol-d 4 at 298 K,
- Figure 29 shows an 1 H-NMR spectra of Propynyi-P[nPrOzi 0 o-b-MeOx 100 ]- methyl 3-mercaptopropionate in methanol-d 4 at 298 K
- Figure 30 shows an 1 H-NMR spectra of Propynyl-P[nPrOzi 10 o-b-MeOx 100 ]- hydroxy in methanol-d 4 at 298 K
- Figure 31 shows the temperature dependent rheology with storage modulus
- Figure 32 shows an 1 H-NMR spectra of Methyl-P[nPrOzi 50 -b-MeOx 50 ]-tert-butyl- piperazine-1 -carboxylat, contaminated with 10% Poly(n-butyl-2- oxazin)
- Figure 33 shows a light microscope image of a printed constructs composed of orthogonal stacks of hydrogel strands with a base area of 12x12 mm 2 and a strand-center to strand-center distance of 3 mm,
- Figure 34 shows cell loaded constructs
- Figure 35 shows results of FACS analysis on the influence of the printing process on the viability of NIH 3T3 fibroblasts.
- MeOx 50 ]-piperidine-4-carboxylic acid ethyl ester represented by curves 1 , 3, 5, 7, 9.
- the elugrams obtained by GPC appear, with the exception of the first batch P1 (curve 1 ), nearly monomodal with only minor tailing to lower molar masses.
- FIG. 2 shows the temperature dependent rheology with storage modulus (G') and loss modulus (G") for 20 wt.-% of Methyl-P[nPrOzi 5 o-b-MeOx 50 ]-piperidine-4- carboxylic acid ethyl ester.
- Storage modulus (G') of batches P1 to P5 is repre- sented by curves 1 1 , 13, 15, 17 and 19 and loss modulus (G") of batches P1 to P5 is represented by curves 21 , 22, 23, 24 and 25.
- thermogelling polymers for which values ⁇ 1 kPa are more commonly found in the literature (C. Li, N. J. Buurma, I. Haq, C. Turner, S. P. Armes, V. Castelletto, I. W. Hamley, A. L. Lewis, Langmuir 2005, 21, 1 1026-1 1033; S. Xuan, C.-U. Lee, C. Chen, A. B. Doyle, Y. Zhang, L Guo, V. T. John, D. Hayes, D. Zhang, Chem. Mater. 2016, 28, 727-737).
- hydrogels of F127 at 20 wt.-% approximately 10 kPa
- G. Grassi A prominent exception are hydrogels of F127 at 20 wt.-% (approximately 10 kPa) (G. Grassi, A. Cre- vatin, R. Farra, G. Guarnieri, A. Pascotto, B. Rehimers, R. Lapasin, M. Grassi, J. Colloid Interface Sci. 2006, 301, 282-290).
- thermogelling polymers are quite distinct from F127 and P123, which also form gels at elevated temperature and/or concentration and are com- monly used for gel plotting in biofabrication (N. E. Fedorovich, J. R. de Wijn, A. J. Verbout, J. Alblas, W. J. A. Dhert, Tissue Eng. Part A 2008, 14, 127-133).
- the viscosity of the new material at low temperature is relatively low, in particular compared to the viscosity of Pluronic ® block copolymers (compare 700 mPa * s (F127) vs. 7 mPa * s (P2) at 10 wt.-% and 10 °C). Even at 30 wt.-%, a solution of P2 at 10 °C (curve 49 in Figure 1 1 ) has a lower viscosity (approx. 50 %) than a 10 wt.-% solution of F127 (curve 51 in Figure 1 1 ), which never forms a gel at this concentration. Based on these rheological experiments, it is possible to sketch a preliminary phase diagram for the new thermoresponsive materials.
- Figure 14 shows the resulting correlation length ⁇ (curve 63) and characteristic domain size d (curve 65) of Methyl-P[nPrOzi 5 o-b-MeOx 50 ]-piperidine-4-carboxylic acid ethyl ester.
- the correlation length is a cutoff length, above which correlations are no longer noticeable in the system. As temperature increases, an increase in the domain size as well as in the correlation length was observed, the latter eventually exceeding the former (at approximately 18 °C).
- Figure 15 shows the representative fits for a bi-continuous sponge like structure of selected datasets over the complete temperature range of Methyl-P[nPrOzi 5 o-b- MeOx 50 ]-piperidine-4-carboxylic acid ethyl ester.
- SANS scattering data and their respective fits were shifted using a factor of 1 (6.9 °C, curve 53), a factor of 4 (21 .1 °C, curve 55), a factor of 16 (30.0 °C, curve 57), a factor of 64 (36.2 °C, curve 59) and a factor of 256 (39.7 °C, curve 61 ), respectively.
- Figure 17 shows the cell viability of NIH 3T3 fibroblasts as a bar chart 81 .
- the bar chart 81 shows results for PI staining 83 with a bar 85 for the control sample, a bar 87 for the methanol treated sample and a bar 89 for the sample with cells in 25% of gel.
- the bar chart 81 shows results for FDA staining 91 with a bar 93 for the control sample, a bar 95 for the methanol treated sample and a bar 97 for the sample with cells in 25% of gel.
- Figure 18 shows a flow cytometry analysis (FACS Calibur system) of NIH 3T3 fibroblasts cultivated in Methyl-P[nPrOzi 5 o-b-MeOx 50 ]-piperidine-4-carboxylic acid ethyl ester, using a 488 nm laser with the emission channel FL2 (585 nm / ⁇ 21 nm) for PI staining 83 (compare to Figure 17) with curve 99 for control sample (see bar 85 in Figure 17), curve 103 for the methanol treated sample (see bar 87 in Figure 17) and curve 101 for the sample with cells in 25% of gel (see bar 89 in Figure 17).
- a total number of 5000 events were counted with BD CellQuestTM Pro and the geometric mean fluorescence intensity was determined for each condition using Flowing Software (version 2.5.1 ; Turku Bioimaging).
- Figure 19 shows a flow cytometry analysis of NIH 3T3 fibroblasts cultivated in Me- thyl-P[nPrOzi 5 o-b-MeOx 50 ]-piperidine-4-carboxylic acid ethyl ester for the emission channel FL1 (530 nm / ⁇ 15 nm) for FDA staining 91 (compare to Figure 17) with curve 105 for control sample (see bar 93 in Figure 17), curve 107 for the methanol treated sample (see bar 95 in Figure 17) and curve 109 for the sample with cells in 25% of gel (see bar 97 in Figure 17).
- FIG 20 a temperature-dependent rheological analysis of Methyl-P[nPrOzi 5 o-b- MeOx 50 ]-piperidine-4-carboxylic acid ethyl ester at a concentration of 20 wt.-% (batch P2) is shown.
- Curve 1 1 1 shows the temperature-dependence of the storage modulus (G')
- curve 1 13 shows the temperature-dependence of the loss modulus (G").
- the gel point, determined at the intersection of G' and G" is located at 35 °C, so just below body temperature. The gel point moves to lower temperatures with increasing the molar mass.
- the resulting hydrogels are quite soft with a loss modulus (G') of around 4 kPa. In contrast to the gelling temperature, the storage modulus (G') seems to be independent of the molar mass.
- Figure 21 shows a phase diagram 1 15 of aqueous solutions of Methyl-P[nPrOzi 50 - b-MeOx 50 ]-piperidine-4-carboxylic acid ethyl ester dependent on concentration and temperature. Increasing temperature, at concentrations below 1 0 wt.-% muddy solutions are obtained, which can be explained by nano- and microscaling aggregation.
- Figure 22 shows the temperature dependent rheology with storage modulus (G') and loss modulus (G") for 20 wt.-% of Methyl-P[nPrOziioo-b-MeOx 100 ]-piperidine-4- carboxylic acid ethyl ester (batch P1 a) and Methyl-P[nPrOzi 10 o-b-MeOx 100 ]- tert- butyl-piperazine-1 -carboxylate (batch P2a).
- Storage modulus (G') and loss modulus (G") of batch P1 a are represented by curves 1 17, 1 19 and storage modulus (G') and loss modulus (G") of batch P2a are represented by curves 121 , 123. It can be seen, that the use of 1 -BOC Piperazine (curves 121 , 123) instead of ethyl- 4-piperidinecarboxylate (curves 1 17, 1 19) as a termination molecule does no influence the gelation behavior of the block copolymer. The storage modulus G' as well as loss modulus (G") remain unchanged.
- Figure 23 shows the temperature dependent rheology with storage modulus (G') and loss modulus (G") for 20 wt.-% of Pro- pynyl-P[nPrOzi 0 Q-b-MeOx 100 ]-piperidine-4-carboxylic acid ethyl ester (batch P3a),
- FIG 24 shows the GPC traces of batch P1 a (curve 137) and batch P2a (curve 139) measured in HFIP.
- the GPC elugrammes 137, 139 have a moderately narrow molecular weight distribution ((D ⁇ 1 ,5) and also show no differences as a result of the used termination molecule.
- Figure 25 shows the GPC traces of batch P3a (curve 141 ), batch P4a (curve 143) and batch P5a (curve 145) measured in DMF. These polymers were used for the preparation of the gels, which are characterized by the temperature-sweep shown in Figure 23.
- Figures 26 to 30 show the 1 H-NMR spectra of batches P1 a ( Figure 26), P2a (Figure 27), P3a ( Figure 28), P4a (Figure 29) and P5a ( Figure 30) in methanol-d 4 at 298 K each.
- the 1 H-NMR spectra 26, 27, 28, 29, 30 of all block copolymers show that there are no detectable rests of solvent, which may influence the gelling process (the formation of the gel) of the respective block copolymer.
- the desired degree of polymerization could be determined by endgroup analysis.
- Figure 31 shows the temperature dependent rheology with storage modulus (G') and loss modulus (G") for 20 wt.-%of contaminated Methyl-P[nPrOzi 5 o-b-MeOx 50 ]- tert-butyl-piperazine-1 -carboxylat (batch P6a).
- the storage modulus (G') is represented by curve 147 and the loss modulus (G") is represented by curve 149.
- Figure 32 shows an 1 H-NMR spectra of batch P6a.
- the Poly(2-n-propyl-2-oxazin)-block was deliberately contaminated with 10% Poly(n-butyl-2-oxazin).
- the resulting block copolymers are opaque, but exhibit storage modulus (G') that is increased by a factor of 2 (curve 147).
- G' storage modulus
- the material can store more deformation energy and influences the effect of reverse deformation. Due to the more pronounced elastic character, a higher degree of cross-linking can be assumed. In this context, it must be taken into account, that it is exclusively a reversible physical cross-linking reaction.
- Figure 33 shows a light microscope image of a printed constructs composed of orthogonal stacks of hydrogel strands.
- the cell distribution within the constructs was homogeneous throughout the entire constructs.
- the homogenous cell distribution was facilitated due to the
- thermoresponsive properties of the material At low temperatures (ice bath) the ink has a very low viscosity and cells are readily distributed within the material via repeated mixing by pipetting. Once taken of the ice, the immediate, temperature driven viscosity increase preserved the homogenous cell distribution within the ink until the material was dispensed.
- Malda et al. it can be challenging to homogeneously distribute cells in highly viscous bioinks due to various issues (air bubbles, difficult pipetting/handling) (V. H. M. Mouser, F. P. W. Melchels, J. Visser, W. J. A. Dhert, D. Gawlitta, J. Malda, Biofabrication 2016, 8, 35003).
- NIH-3T3 cells included into biofabricated scaffolds were further investigated via flow cytometry.
- the results can be taken out of Figure 35. While the untreated control represents cells in medium, the control represents cells that were dispersed in the bioink but not printed. This revealed similar levels of cytocompatibility (91 .5 % ⁇ 0.8 % - bar 159) compared to cells incorporated into the material without further processing (92.8 % ⁇ 1 .7 % - bar 161 ) and untreated control cells (98.9 % ⁇ 0.18 % - bar 163) Therefore, the printing process seems to have no effect on the cell viability when using our bioink.
- thermogelling synthetic block copolymers comprising a hydrophilic block [A] or [B] and a thermoresponsive block [A] or [B], which are an excellent bioink candidates.
- the new gels are optical transparent and have a very suitable and adjustable gelling temperature.
- the synthesis of the polymers is easy and to be scaled well.
- the gelation process of all describes molecules is very fast.
- the combination of thermogelation, excellent biocompatibility and isothermal shear-thinning is particularly attractive for many applications including drug delivery, biofabrication, cell culture or tissue engineering.
- the particularities of the rheological properties can be conveniently fine-tuned via the polymer composition while the chemical functionalization via chain termini can be realized without having an impeding influence on the desirable rheological properties.
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